Fig. 4. Dominant-negative Rac1 and inhibitors of RhoA and ROCK block FHOD1 C-dependent stress fibers. (A) Rac1N17 blocks FHOD1-C-dependent stress fibers. NIH-3T3 cells were transiently transfected with pCMV5-FHOD1 C and pCMV-T7-Rac1N17. FHOD1- and RacN17-expressing cells were detected by in situ immunofluorescence. F-actin was detected with phalloidin-rhodamine. (B) RhoA activity is required for FHOD1-C-dependent stress fibers. FHOD1-C-transfected NIH-3T3 cells were incubated for 18 hours with 10 µM or 30 µM C3 transferase prior to in situ fluorescence analysis and confocal microscopy. (C) ROCK activity is required for FHOD1-C-dependent stress fiber formation. FHOD1-C-transfected NIH-3T3 cells were incubated for 10 minutes with 30 µM Y-27632. FHOD1 C and F-actin were detected with in situ fluorescence and confocal microscopy.